The field of this invention is the area of plant molecular biology, and it relates in particular to transcription regulatory elements: a qualitative regulatory sequence which positively regulates downstream gene expression in plant tissue in response to the stress of an invading microbial pathogen, an elicitor, or other inducing chemical signals and quantitative regulatory sequences which increase the transcriptional expression of associated sequences.
In plants, disease resistance to fungal, bacterial and viral pathogens is associated with a plant response termed the hypersensitivity response (HR). In the HR, the site in the plant where the potential phytopathogen invades undergoes localized cell death, and it is postulated that this localized plant cell death aspect of the HR contains the invading microorganism or virus, thereby protecting the remainder of the plant. Other plant defenses include the production of phytoalexins (antibiotics) and/or lytic enzymes capable of averting pathogen ingress and/or cell wall modifications which strengthen the plant cell wall against physical and/or enzymatic attack.
The HR of plants, including tobacco, can include phytoalexin production as part of the response to invading microorganisms. One class of compounds made by tobacco (Nicotiana tabacum) in response to microbial invaders are the sesquiterpenes.
Cell suspension cultures have provided useful information regarding the regulation of terpene synthesis. Isoprenoids are ubiquitous in nature, and the early portions of the biosynthetic pathway are shared with the biosynthetic pathway for other isoprenoid compounds such as sterols, carotenoids, dolichol and ubiquinone and growth regulators (e.g., gibberellic acid), which are classified as primary metabolites. Isoprenoid compounds classified as secondary metabolites are not essential for growth, and include mono-, sesqui- and diterpenoids. These secondary metabolite isoprenoids are important mediators of interactions between the plant and its environment.
A variety of compositions can serve as elicitors of plant phytoalexin synthesis. These include, but are not limited to, one or more toxic ions, e.g., mercuric ions, other chemically defined compositions, metabolic inhibitors, cell wall glycans, certain glycoproteins, certain enzymes, fungal spores, chitosans, certain fatty acids and certain oligosaccharides derived from plant cell walls [See, e.g., Sequeira, L. (1983) Annu. Rev. Microbiol. 37:51-79 and references cited therein]. Epi-5-aristolochene synthase (EAS) activity in tobacco plants has been shown to be induced by cell wall fragments of certain Phytophthora species and by Trichoderma reesei cellulase but not Aspergillus japonicum pectolyase [Chappell et al. (1991) Plant Physiol. 97:693-698]. Attack by other plant pathogens or an avirulent related strain can also induce phytoalexin synthesis; for example, Pseudomonas lachrymans induces sesquiterpenoid synthesis in tobacco [Guedes et al. (1982) Phytochemistry 21:2987-2988].
Elicitins are proteins which are produced by plant pathogens and potential plant pathogens, which proteins induce the HR in tobacco plants. Amino acid and nucleotide coding sequences for an elicitin of Phytophthora parasitica have been published [Kamoun et al. (1993) Mol. Plant-Microbe Interactions 6:573-581]. Plant pathogenic viruses including, but not limited to, Tobacco Mosaic Virus (TMV) induce the HR in infected plants. Bacteria which infect plants also can induce HR and thereby disease resistance; representative bacteria eliciting HR include, but are not limited to, Agrobacterium species, Xanthomonas species and Pseudomonas syringae. Plant pathogenic fungi (and certain avirulent strains as well) also induce the HR response, where these include, but are not limited to, Phytophthora parasitica and Peronospora tabaci. 
When tobacco cell suspension cultures are treated with an elicitor, squalene synthetase is suppressed, thus stopping the flow of common biosynthetic precursors into sterols. The concomitant induction of sesquiterpene cyclase gene expression causes the flow of precursors in sesquiterpenes. The first step in the pathway from farnesyl diphosphate to the sesquiterpene phytoalexin capsidiol in elicitor-induced tobacco tissue is catalyzed by 5-epi-aristolochene synthase (EAS), a sesquiterpene cyclase. The coding sequence and deduced amino acid sequence for one member of the EAS gene family of tobacco have been published [Facchini and Chappell (1992) Proc. Natl. Acad. Sci. USA 89:11088-11092]. The transcriptional expression of one or more members of the EAS gene family is induced in response to elicitors.
There is a long felt need in the art for methods of protecting plants, particularly crop plants, from infection by plant pathogens, including but not limited to, phytopathogenic viruses, fungi and/or bacteria. Especially important from the standpoint of economics and environmental concerns are biological or xe2x80x9cnaturalxe2x80x9d methods rather than those which depend on the application of chemicals to crop plants. There is also a long felt need in the art for plant transcriptional regulatory sequences for use in controlling the expression of heterologous DNA sequences in transgenic plants.
The present invention provides qualitative transcriptional regulatory sequences which regulate downstream gene expression in plant tissue in response to one or more elicitors, other defined inducing compounds or in response to the stress of an invading phytopathogen (the inducible transcription regulatory sequence) and quantitative transcription regulatory sequences which increase the transcription of downstream sequences (the transcription-enhancing sequence). As specifically exemplified herein, these transcriptional regulatory sequences are found in nature upstream and operatively linked to the epi-5-aristolochene synthase gene (EAS4) of tobacco; when operatively linked to a coding sequence (and in the presence of an operatively linked promoter element, from the EAS4 gene or from a heterologous plant-expressible gene) these sequences mediate the inducible transcriptional expression of that coding sequence when the plant or plant tissue is invaded by a potential phytopathogen (virus, fungus or bacterium) or in response to elicitors such as Trichoderma viride cellulase or plant or fungal cell wall fragments for plants, plant tissue and/or plant suspension culture cells. That potential plant pathogen can be a virus including, but not limited to, tobacco mosaic virus or tobacco vein mottle virus, a bacterium including, but not limited to, Pseudomonas syringae, Xanthomonas campestris or Agrobacterium tumefaciens, or a fungus including, but not limited to, a species of Phytophthora (e.g., P. parasitica) or Peronospora (e.g., P. tabaci). The EAS4 promoter comprising the inducible transcription regulatory element(s) and the transcription-enhancing sequence(s) are disclosed herein as SEQ ID NO:2. In SEQ ID NO:2, the CAAT-homologous sequence of the EAS4 promoter is located at nucleotides 513 to 516, and the TATA-sequence motif is located at nucleotides 540 to 543.
The minimal inducible transcriptional regulatory element within the N. tabacum EAS4 upstream sequence is from nucleotide 458 to nucleotide 473 of SEQ ID NO:2; optionally from 454 to 473; more preferably from nucleotide 413 to 473 in SEQ ID NO:2 provides the inducible transcriptional regulatory element sequences.
Another aspect of the present invention is the transcription-enhancing element derived from the EAS4 promoter and promoter-associated sequences. When operatively linked upstream of a promoter, particularly upstream of a minimal promoter, this element increases the transcriptional expression of the downstream sequences. Transcriptional enhancing activity is mediated by DNA sequence information in the region between nucleotides 371 and 463 in SEQ ID NO:2. Preferably, an EAS4-derived transcription-enhancing sequence comprises a nucleotide sequence as given in SEQ ID NO:2 from nucleotide 371 to nucleotide 463, more preferably from nucleotide 1 to nucleotide 463, and optionally from nucleotide 1 to about nucleotide 1040 of SEQ ID NO:7.
Also provided by the present invention is an expression cassette into which a coding sequence of interest can be cloned, and said coding sequence of interest can be expressed in plant tissue after the introduction of the unit into plant tissue. A preferred coding sequence of interest is that for the ParA1 elicitin protein of Phytophthora parasitica. The coding sequence and deduced amino acid sequence for the ParA1 protein, including the signal peptide, are given in SEQ ID NOs:16 and 17, respectively. The coding sequence and deduced amino acid sequences of the nature ParA1 protein as amplified and described hereinbelow are given in SEQ ID NOs:18 and 19, respectively. Transcription is regulated by the EAS4 promoter sequence and promoter-associated regulatory element or by at least one of the transcription regulatory elements (inducible and/or transcription-enhancing) operably linked to transcription initiation sequences and a heterologous DNA sequence to be expressed.
A further aspect of the present invention are transgenic plant cells, plant tissue and plants which have been genetically engineered to contain and express a nucleotide sequence of interest, preferably a coding sequence, an antisense sequence or other sequence, under the regulatory control of the inducible transcription regulatory element. It is an object of this invention to provide the nucleotide sequences which mediate the induction of the expression of a downstream coding sequence in response to elicitor exposure, potential phytopathogen invasion in a plant, or certain other exogenous inducing signals such as exposure to methyl jasmonate and ethylene. An exemplary elicitor inducible transcription regulatory element is that from the 5xe2x80x2 flanking region of the EAS4 gene of Nicotiana tabacum; as specifically exemplified herein, this sequence is presented in SEQ ID NO:2 from nucleotide 410 to nucleotide 472. Equivalents of the exemplified nucleotide sequence are those nucleotide sequences which similarly direct the induction of the expression of downstream nucleotide sequences. Preferably the inducible transcription regulatory element is associated with EAS4 promoter and promoter-associated sequences (e.g., the combination having the nucleotide sequence as given in SEQ ID NO:2 from nucleotide 410 to nucleotide 573 of SEQ ID NO:2, preferably from nucleotide 361 to 573 of SEQ ID NO:2, more preferably the sequence from 1 to 573 of SEQ ID NO:2).